Administration of biologically active vitamin D3 and vitamin D2 materials

ABSTRACT

A process for administering biologically active vitamin D 3  and vitamin D 2  materials into the blood system of a subject by providing the biologically active vitamin D 3  or D 2  material to the skin whereby the skin releases the biologically active vitamin D 3  or D 2  material into the blood system in a controlled and prolonged manner.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 137,673, filed Apr. 4, 1980, now abandoned, whichin turn is a divisional application of Ser. No. 22,393, filed Mar. 21,1979, now U.S. Pat. No. 4,230,701 issued Oct. 28, 1980.

BACKGROUND OF THE INVENTION

Biologically active vitamin D₃ and vitamin D₂ materials, due to theiraction in increasing serum calcium have been utilized in the treatmentof various calcium metabolic disorders. Among these disorders includechronic renal disease, Vitamin D resistant rickets,glucocorticord-induced decrease in calcium absorption, osteoporosis,senile decrease in calcium absorption, hypoparathyroidism, turkey weakleg diseases, milk fever diseases, and the like. In the past,biologically active Vitamin D₃ and Vitamin D₂ materials have beenadministered to patients orally or intravenously.

A danger in administering biologically active viatmin D₃ and vitamin D₂materials orally or intravenously to patients is that the therapeutic totoxic ratio (therapeutic index) of this drug is low and an exess of thedrug in the blood stream, especially after administration, can causeepisodes of hypercalcemia and hypercalcuria. While hypercalcemia andhypercalcuria can be corrected to a large degree by decreasing the doseof the drug, there is always the danger of hypercalcemia andhypercalcuria repeatedly occuring each time a patient receives a givenoral or intravenous dose. This is believed due to the fact thatimmediately after administration of these drugs, the drug is transportedin high concentrations across the small intestine which is one of theprincipal sites of activity for these drugs. Thus, especially after oraladministration, the small intestine is initially exposed to very highconcentrations of these metabolites which in turn could induce a veryrapid uncontrolled rise in intestinal calcium transport which isreflected in high serum calcium levels in the blood.

Therefore, it has been desired to provide a means for administeringbiologically active vitamin D₃ or vitamin D₂ materials to avoid theuncontrolled increase in serum calcium levels which could occurimmediately after administration of the biologically active vitamin D₃and vitamin D₂ materials.

SUMMARY OF INVENTION

In accordance with this invention, it has been found that whenbiologically active vitamin D₃ and vitamin D₂ materials are administeredfor therapeutic purposes on or into the skin of a subject, the skin actsas a transport medium for the biologically active vitamin D₃ and vitaminD₂ materials. By acting as a transport mechanism whereby these materialsare allowed to enter the blood stream of the circulatory system of thesubject, these biologically active vitamin D₃ or vitamin D₂ materialsare transported into the blood stream in a slow and controlled manner.Through this mechanism, the biologically active vitamin D₃ and vitaminD₂ materials are administered to patients without a rapid anduncontrolled rise in the serum calcium levels which occur duringadministration by oral and injection means.

Furthermore, when these biologically active vitamin D₃ and vitamin D₂materials are provided to the skin of subjects through application on orinto the skin, the rise in the intestinal calcium response and in theserum calcium levels induced by a single application can last for atleast two weeks after application while a single oral dose of the samequantity is substantially effective for about seven days. Hence, by theprocedure of this invention, the need for several repeated doses of thebiologically active vitamin D₃ and vitamin D₂ materials for controllingcalcium levels in a given period is substantially reduced therebyreducing the danger of the uncontrolled and rapid rise of serum calciumlevel which would occur after each administration of the dose to causehypercalcemia and hypercalcuria.

The process of this invention is carried out by providing to the skin ofthe subject to be treated a biologically active vitamin D₃ or D₂material and allowing this biologically active material to transportthrough the skin into the blood stream. In carrying out the process ofthis invention, any conventional method of providing a biologicallyactive vitamin D₃ or vitamin D₂ material to the skin can be utilized.Once the biologically active vitamin D₃ or vitamin D₂ materials areapplied to the skin, the skin will act as a means for storing andtransporting the biologically active materials into the circulationsystem of the subject. The skin acts as a vehicle for transport of thebiologically active materials to the blood system in such a manner thatit is only released onto the blood system in such a controlled mannerthat the rise in the serum calcium level is regulated thereby preventinga rapid uncontrolled rise. Furthermore, the skin acts as a means forstoring the biologically active vitamin D₃ or vitamin D₂ active materialso that one administration of the biologically active vitamin D₃ orvitamin D₂ materials to the skin can regulate the serum calcium levelfor at least two weeks.

DETAILED DESCRIPTION

In accordance with this invention, any conventional method of providinga biologically active vitamin D₂ or D₃ material to the skin of a subjectcan be utilized. Among the conventional methods is by applying to theskin the biologically active vitamin D₂ or D₃ material are includedapplying this material topically intraepidermally, intradermally or intothe subcutaneous fat.

The vitamin D₂ or D₃ material can, in accordance with one embodiment ofthis invention, be applied to the skin topically. In accordance withthis embodiment, the biologically active vitamin D₂ or D₃ material canbe applied to the skin either as the biologically active vitamin D₂ orD₃ material itself or as the corresponding previtamin or correspondingprovitamin. Any conventional method of topical application orconventional topical preparation can be utilized to apply either theprovitamin or previtamin of the biologically active vitamin D₃ or D₂material or even the biologically active D₂ or D₃ material itself to theskin. In the case where the biologically active vitamin D₃ or vitamin D₂material is provided to the skin by the provitamin of the biologicallyactive vitamin D₂ or D₃ materials, the provitamin is applied topicallyto the skin and the skin is subjected to ultra-violet light radiation.The ultra-violet radiation converts the provitamin to the correspondingprevitamin of the biologically active vitamin D₂ or vitamin D₃ material.This previtamin is a relatively unstable material which upon exposure atroom temperature to the skin isomerizes to the biologically activevitamin D₂ or D₃ material. Therefore, the biologically active vitamin D₂or vitamin D₃ material can be provided to the skin by either theprovitamin or previtamin D₃ corresponding to the biologically activevitamin D₂ or vitamin D₃ material. The previtamin corresponding to thebiologically active vitamin D₂ or D₃ material can be topically orintradermally applied to the skin and allowed to isomerize on the skinto the biologically active vitamin D₂ or D₃ material. On the other hand,the biologically vitamin D₂ and vitamin D₃ active material itself can betopically applied to the skin thereby causing the skin to act as atransport medium for releasing the vitamin D₂ or D₃ active material tothe blood system.

Where the biologically active vitamin D₂ and D₃ material is applied tothe skin intradermally, intraepidermally or into the subcutaneous fattissue, it is generally preferred to apply the biologically activevitamin D₂ or D₃ material and its corresponding previtamin. On the otherhand, application of provitamins by these methods to provide thecorresponding previtamin to the skin may require a high degree ofultraviolet light radiation, depending upon the pigmentation of theskin, to convert the provitamin to corresponding biologically activevitamin D₂ or D₃ material. Therefore, it is generally not preferable toapply the vitamin D₂ or D₃ material to the skin, by introducing thecorresponding provitamin intradermally, intraepidermally or into thesubcutaneous fat tissue.

The improved results of this invention are achieved throughadministering the biologically active vitamin D₂ and D₃ materials to theskin of various subjects such as mammals as well as avian species suchas turkeys, chickens, etc. The method of this invention is useful intreating conditions characterized by low serum levels of vitamin D₂ orD₃, especially where these levels result from an inability or an imparedability to convert vitamin D₂ or vitamin D₃ to its biologically activemetabolite. These conditions include among others, chronic renaldisease, vitamin D resistant rickets, glucocorticord-induced decrease incalcium absorption, osteoporosis, senile decrease in calcium absorption,hypoparathyroidism, milk fever disease, turkey weak leg disease, etc.

By administering the biologically active vitamin D₂ or vitamin D₃materials to subjects whose ability to convert vitamin D₂ or vitamin D₃to these biologically active forms is impaired, one is able to providethe necessary biologically active forms of these vitamins to subjects.Therefore, any biologically active form of vitamin D₂ or D₃ can beadministered in accordance with this invention. Among the preferredactive forms of vitamin D₂ and vitamin D₃ which can be administered inaccordance with this invention are the biologically active metabolitesof vitamin D₂ and vitamin D₃ as well as analogues thereof. Thesebiologically active metabolites and analogues contain the vitamin D₂ andD₃ structures and have at least one hydroxy group in addition to thehydroxy group present in the vitamin D₂ or vitamin D₃ structure and mayalso contain a halo substituent. Among the biologically active vitaminD₂ or D₃ material compounds of the formula are preferred: ##STR1##wherein R is methylidene or methyl; R₂ is hydrogen hydroxy or methyl;R₁, R₃ and R₄ are hydrogen, hydroxy or halogen; A and B are individuallyhydrogen or take together form a carbon to carbon bond; with the provisothat at least one of R₁, R₂, R₃ or R₄ is hydroxy and with the furtherproviso that when A and B form a carbon to carbon bond, R₂ is methyl.

The previtamins corresponding to the biologically active vitaminmaterials of formula I have the formula: ##STR2## wherein R₁, R₂, R₃, R₄and A and B are as above with the proviso that at least one of R₁, R₂,R₃ or R₄ is hydroxy and with the further proviso that when A and B forma carbon to carbon bond, R₂ is methyl.

The provitamins corresponding to the biologically active materials havethe formula: ##STR3## where R₁, R₂, R₃, R₄ and A and B are as above withthe proviso that at least one of R₁, R₂, R₃ or R₄ is hydroxy; and withthe further proviso that when A and B form a carbon to carbon bond, R₂is methyl.

In all of the formulae given above, one or more of the hydroxy groupscan be esterified with a lower alkanoyl group containing from 2 to 7carbon atoms such as acetyl.

Among the preferred biological active vitamin D₂ and D₃ materials whichare applied in accordance with this invention are the biological activemetabolites and analogues of vitamin D₂ and vitamin D₃ as well as thecorresponding provitamins and previtamins of these biologically activevitamin D₂ or D₃ metabolites or analogues.

Among the preferred provitamins of these biologically active vitamin D₂or D₃ metabolites or analogues are included:

1α,25-dihydroxy-7-dehydrocholesterol[1α,25-(OH)₂ proD₃ ];

1α,24,25-trihydroxy-7-dehydrocholesterol[1α,24,25-(OH)₃ proD₃ ];

24,25-dihydroxy-7-dehydrocholesterol[24,25-(OH)₂ proD₃ ];

1α-hydroxy-7-dehydrocholesterol[1α-OHproD₃ ];

1α,24-dihydroxy-35-fluoro-7-dehydrocholesterol[1α,24-(OH)₂ 25FproD₃ ];

25,26-dihydroxy-7-dehydrocholesterol[25,26-(OH)₂ proD₃ ];

25-hydroxy-7-dehydrocholesterol[25-(OH)proD₃ ];

1α-hydroxy-ergosterol[1α-OHproD₂ ];

25-hydroxy-ergosterol[25-OHproD₂ ]; and

1α,25-dihydroxy-ergosterol[1α,25-(OH)₂ proD₂ ].

Among the preferred previtamin D₃ metabolites are:

1α,25-dihydroxy-precholecalciferol[1α,25-(OH)₂ preD₃ ];

1α,24,25-trihydroxy-precholecalciferol[1α,24,25-(OH)₃ preD₃ ];

24,25-dihydroxy-precholecalciferol[24,25-(OH)₂ preD₃ ];

1α-hydroxy-percholecalciferol[1α-OHpreD₃ ];

1α,24-dihydroxy-25-fluoro-precholecalciferol [1α,24-(OH)₂ 25FpreD₃ ];and

25-hydroxy-precholecalciferol [25-OHpreD₃ ].

1α-hydroxy-previtamin D₂ [1α-OHpreD₂ ];

25-hydroxy-previtamin D₂ [25-OHpreD₂ ] and

1α,25-dihydroxy-previtamin D₂ [1α,25-(OH)₂ preD₂ ].

The following biologically active vitamin D₂ and D₃ compounds areparticularly preferred:

Dihydrotachysterol₂ ;

Dihydrotachysterol₃ ;

5,6-trans-vitamin D₃ ;

25-hydroxy-5,6-trans vitamin D₃ ;

1α-hydroxy vitamin D₂ [1α-OHD₂ ];

25-hydroxy vitamin D₂ [25-OHD₂ ];

1α,25-dihydroxy vitamin D₂ [1α,25-(OH)₂ D₂ ].

1α,25-dihydroxy-cholecalciferol[1α,25-(OH)₂ D₃ ];

1α,24,25-trihydroxy-cholecalciferol[1α,24,25-(OH)₃ D₃ ];

24,25-dihydroxy-cholecalciferol[24,25-(OH)₂ D₃ ];

1α,24-dihydroxy-25-fluoro-cholecalciferol[1α,24-(OH)₂ 25FD₃ ];

25-hydroxy-cholecalciferol[25-OHD₃ ]; and

1α-hydroxy-cholecalciferol[1α-OHD₃ ]

Among these preferred vitamin D₃ compounds, the vitamin D₃ compoundwhere the 24-hydroxy group has the R configuration is especiallypreferred. Also preferred are those 24,25-dihydroxy-vitamin D₃ compoundswhere the 24-hydroxy group has an R-configuration.

The aforementioned biologically active vitamin D₂ and D₃ materials,their corresponding previtamins and provitamins can be applied to theskin topically or intradermally, intraepidermally or subcutaneousutilizing any conventional method for applying pharmaceuticals in thismanner. In general, one application can contain at least 0.05 microgramsof the active material per administration. Generally, these materialscan be applied in amounts of 0.5 to about 100 micrograms peradministration with 0.1 to 10 micrograms per administration beingpreferred. In general, amounts of these biologically active vitamin D₂or D₃ materials, i.e. either as the previtamin, vitamon or provitamincan be applied typically in doses of greater than 100 micrograms peradministration. This is especially true since through the application ofthese materials to the skin, the amount of the biologically activevitamin D₃ or D₂ materials transported to the bloodstream is regulatedand stored in the skin. Therefore, the danger of applying thebiologically active vitamin D₃ or D₂ materials in excessive dosages isminimized through the controlled transport by the skin to thebloodstream.

When provitamin D₃ corresponding to the biologically active vitamin D₂or D₃ material is applied to the skin, the provitamin is converted tothe corresponding previtamin by the exposure to ultraviolet light. Thisultraviolet light can be the sun or can be supplied through aconventional ultraviolet light lamp. Generally when a ultraviolet lightlamp is utilized and the provitamin supplied topically to the skin, 0.1to 10 joules per square centimeter per second is applied. The light canbe applied for a fraction of a second to ten hours or longer. The amountof light supplied is not critical. The previtamin which is formed on theskin is isomerized to the biologically active vitamin D₂ or D₃ materialby exposure to the skin at room temperature. The skin provides the meansfor thermally isomerizing the previtamin to the biologically activevitamin D₂ or D₃ material.

The biologically active vitamin D₂ and D₃ materials as well as theirprevitamins and provitamins, can be applied to the skin throughconventional methods. Any conventional topical subcutaneous orintradermal preparation can be utilized to apply these materials to theskin. Any of the means conventional in applying pharmaceuticals intopical subcutaneous or intradermal forms with conventionalpharmaceutical carriers can be utilized in accordance with thisinvention.

For topical administration, the biologically active vitamin D₂ and D₃materials as well as their previtamins and provitamins can beconventionally prepared as ointments, tinctures, creams, gels,solutions, lotions, sprays, suspensions and the like. Ointments, creamsand solutions are preferred. The pharmaceutical preparation for topicaladministration can be prepared by mixing the aforementioned activeingredients with non-toxic, therapeutically inert, solid, or liquidcarriers which are customarily utilized in such preparations. Whenprepared as solutions, the weight concentration of the active ingredientis about from 0.001% to about 7.0%, preferably from 0.02% to 0.5%. Whenprepared in the form of ointments and creams, the weight concentrationof the aforementioned active ingredients are about from 0.001% to about7.0%, preferably from 0.05% to about 1%. In preparing compositionscontaining the previtamin of the biologically active vitamin D₂ or D₃material, care must be exercised to keep the material under conditionsof temperature, i.e. below room temperature to impede its conversion tothe biologically active vitamin D₂ or D₃ material. Furthermore, theprevitamin should be applied almost after preparation in view of itsinstability.

It is contemplated to incorporate into the topical preparationsdescribed above additives such as preservatives, thickeners, perfumesand the like which are recognized as being conventional in the art ofpharmaceutical compounding. In addition, it is contemplated toincorporate into the topical preparations herein described one or amixture of conventional antioxidants such as, for example,N-methyl-α-tocopherolamine, tocopherols, butylated hydroxyanisole,butylated hydroxy-toluene, ethoxyquin and the like.

This invention thus also relates to the use of pharmaceuticallyacceptable cream and ointment compositions of the vitamin D₂ or D₃active materials as well as their corresponding pre or provitamins.

Cream base pharmaceutical formulations acceptable for use with vitaminD₂ or D₃ active materials as well as their corresponding pre orprovitamins comprise aqueous emulsions containing a fatty acid alcohol,a semisolid petroleum hydrocarbon, a 1,2-glycol and an emulsifyingagent.

Ointment pharmaceutical formulation acceptable for use with vitamin D₂or D₃ active materials as well as their corresponding pre or provitaminscomprise admixtures of a semi-solid petroleum hydrocarbon with a solventdispersion of the active material.

Cream composition of the vitamin D₂ or D₃ active materials as well astheir corresponding pre or provitamins, preferably comprise emulsionsformed from a water phase of a humectant, a viscosity stabilizer andwater, an oil phase of a fatty acid alcohol, a semi-solid petroleumhydrocarbon and an emulsifying agent and a phase containing the activeingredient dispersed in an aqueous stabilizer-buffer solution.

The water phase comprises, as the humectant, a pharmaceuticallyacceptably polyhydric alcohol as, for example, a 1,2-glycol of theformulat RCHOHCH₂ OH wherein R is an alkyl of from 1 to 5 carbon atoms.A particularly effective polyhydric alcohol, and the preferred one ofthis invention, is propylene glycol. Although the polyhydric alcoholshelp to maintain active ingredients in the emulsion, their concentrationtherein is not critical, an effective concentration being from about 5to about 20% by weight with a preferred concentration being from about12 to about 15% by weight.

A methyl cellulose or hydroxypropylmethyl cellulose may be added tostabilize the viscosity of the cream during storage. Particularlyeffective is a hydroxypropylcellulose known as Methocel 65 HG 4000 fromDow Chemical. The effective concentration range is from about 0.1 toabout 5% by weight with from about 0.5 to about 0.8% preferred.

In the oil phase, the fatty acid alcohol component, which functions as astabilizer, is derived from the reduction of a long-chain saturatedfatty acid of from about 14 to about 20 carbon atoms as, for example,stearyl and cetyl alcohol. The effective concentration is from about 10to about 30% by weight with an optimum concentration of from about 15 toabout 20% by weight. Stearyl alcohol is the preferred component.

The semi-solid petroleum hydrocarbon emollient of the oil phase is apurified mixture of hydrocarbons of the methane series having theformula CnH2n+2 and known, in general, by the names petrolatum,petroleum jelly or vaselin. These materials have a melting range of fromabout 34° to about 54° C., a density range of from about 0.820 to about0.870 and a refractive index range of from about 1.460 to about 1.474.The preferred material is Petrolatum Perfecta, a purified mixture ofsemi-solid hydrocarbons (C₂₀ -C₂₂) from petroleum, melting point range38°-50° C. It is used in concentrations of from about 5 to about 40% byweight with from about 10 to about 15% by weight preferred.

Finally, the oil phase can optionally contain water-soluble emulsifyingagents such as polyoxyethylene glycol and polyoxypropylene glycol, usedin effective concentrations, i.e., from about 5 to about 8% by weight.

The vitamin D₂ or D₃ active materials as well as their corresponding preor provitamins either as prepared or subsequently micronized issufficiently stable in the excipients used in formulations to providethe concentration needed for therapeutic use.

The invention is illustrated by the following examples. In the examples,Sephadex LH-20 is a hydroxy-propyl ether derivative of a polydextrangel. Nedox 1518 Sephadex LH-20 is Sephadex LH-20 modified to contain C15through C18 aliphatic saturated chains linked by an ether linkage to thepolydextran chain of Sephadex LH-20 prepared as disclosed by Ellingboeet al. J. Lipid Res. 11:266-273 (1940). The everted gutsac techniqueutilized in these examples is described by Martin and DeLuca, Arch.Biochem. Biophys. 134:139-148 (1969). In the Examples, the intestinalcalcium transport is given by the ratio of the calcium concentrationoutside the gutsac (O) to the concentration of calcium inside theinverted gutsac (I) as set forth in the Martin and DeLuca article. Theserum calcium determination utilized in the Examples is described byTanaka et al. Arch. Biochem. Biophys. 146:574-578 (1971).

EXAMPLE 1 Synthesis [24-tritiated]-1α,25-dihydroxy-7-dehydrocholesterol

Cholesta-24-ene-1α,3β-diacetate (0.1 g) was dissolved in 3 cctetrahydrofuran (THF) and 2 cc H₂ O. The mixture was cooled in an icebath and 70 mg mercuric acetate was added all at once. After 30 min thereaction was removed from the ice bath and the reaction was continuedfor an additional 24 h at 25° C. The reaction was extracted withdiethylether (Et₂ O) and H₂ O and the ether phase was collected and asmall portion applied to a silica gel thin layer plate. The plate wasdeveloped in 8:2 (v/v) n-hexane/ethylacetate and indicated that greaterthan 80% of the starting material (R_(f) 0.7) was converted to thedesired mercuryl product, i.e. 1α,3β diacetoxy-25-hydroxy-24mercuric-cholesterol (R_(f) 0.3). The product (50 mg) was dissolved in 3cc THF and 1 cc H₂ O and while stirring under N₂ 3.36 mg[tritiated]-sodium borohydride S.A. 11.2 Ci/mmole was added. After 30min at room temperature the reaction was extracted with Et₂ O/H₂ O theether phase was collected and the H₂ O phase was extracted 2× with Et₂O. The Et₂ O phases were combined, dried under N₂ and dissolved in 1 ccof 19:1 n-hexane/CHCl₃ (v:v) and applied to a 1×60 cm glass columncontaining 15 g of Nedox 1518 Sephadex LH-20 that was slurried, packedand run in the same solvent. Fractions (2 ml) obtained by utilizing the19:1 n-hexane/CHCl₃ (v:v) solvent, were collected and the productcontaining 24-tritiated 1α,3β-diacetoxy-25-hydroxy-cholesterol waslocated in fractions 20 through 30. Fractions 20 through 30 werecombined dried under N₂ and the residue 25 mg was dissolved in 5 ccacetic anhydride containing 0.2 cc pyidine and refluxed. After 24 hr thereaction was dried under N.sub. 2, redissolved in 0.3 cc 19:1n-hexane/CHCl₃ (v:v) and applied to a 1×60 cm glass column containingNedox 1518 Sephadex LH-20. The product was collected dried under N₂ andthe residue (30 mg) of 24-tritiated-1α,3β,25-triacetoxy-cholesterol wasdissolved in 5 cc 1:1 n-hexane/benzene (v:v) and warmed to 72° C. in aH₂ O bath. 1,3-dibromo-5,5'-dimethylhydantoin (10 mg) was added andafter 30 min at 72° C. the reaction was filtered. The filtrate was driedunder N₂ redissolved in 2 cc xylene and added to a mixture of 0.2 cctrimethylphosphite in 3.0/cc xylene that was maintained at 135° C. After1.5 hr the reaction was dried under N₂ redissolved in 10 cc 19:1 MeOH/H₂O containing 50 mg KOH and refluxed for 24 hr. The reaction wasextracted with CHCl₃ /H₂ O, the CHCl₃ phase collected while the H₂ Ophase was extracted x2 with CHCl₃. The CHCl₃ phases were combined driedunder N₂ redissolved in 1 cc 35:65 n-hexane/CHCl₃ (v/v) and applied to a1×60 cm glass column containing 15 g Sephadex LH-20 that was slurriedand packed in the same solvent. Fractions (3 ml) were obtained byeluting the column with 35:65 n-hexane/CHCl₃ (v:v) and collected. Thecompound 24-tritiated-1α,25-dihydroxy-7-dehydrocholesterol was obtainedin fractions 90 through 120. This compound was identified by UVadsorption spectra and its ability to co-elute with an authentic sample.

EXAMPLE 2 Topical application of [24-Tritiated]-1α,25-(OH)₂-7-dehydrocholesterol on rats

Groups of weanling rats that were maintained on a normal calcium andnormal phosphorus vitamin D-deficient diet for 3 wks had their backsshaved. 24 h later 1-2μ Ci [24-tritiated]-1α,25-(OH)₂-7-dehydro-cholesterol in 0.1 ml of Wesson oil was topically appliedover a 9 cm² shaved area and 2 h later groups of animals were eitherleft in the dark or exposed to ultraviolet irradiation under a sun lamp(spectral range 253-400 nm) for a total dose of 0.23 joules/cm² atλ_(max) 297 nm. Immediately after irradiation the animals in both groupswere exsanguinated and their skins were removed and frozen on dry ice.The skins were minced and homogenized for 30 sec. The homogenates wereextracted with CHCl₃ :MeOH and the CHCl₃ phase was collected, driedunder N₂ at 0° C. and applied to a 1×60 cm Sephadex LH-20 column packedin and developed with 65:35 CHCl₃ /n-hexane (v/v). Chromatograms of thelipid extracts from rat skins, small intestines and from the blood ofthe animals that either remained in the dark or received 0.23 joules/cm²of ultraviolet irradiation after receiving [24-tritiated] 1α,25-(OH)₂-7-dehydrocholesterol were carried out. With respect to the rats whichreceived ultraviolet radiation both 1α,25-(OH)₂ -7-dehydrocholesteroland 1α,25-(OH)₂ -cholecalciferol were found in the lipid extracts fromthe skins, the small intestine and the blood of the rats. On the otherhand, only 1α,25-(OH)₂ -7-dehydrocholesterol and no trace of 1α,25-(OH)₂-cholecalciferol was detected in the same lipid extracts from the skins,small intestine and the blood of the rats which were kept in the dark.

EXAMPLE 3 Topical application of[23,24-tritiated]25-hydroxy-7-dehydrocholesterol on rats

Four rats, which had been on a vitamin D-deficient diet that wasadequate in calcium and phosphorus for 21/2 weeks, were shaved and 24hours later were topically dosed over a 9 cm² area with[23,24-tritiated]-25OH-7-dehydrocholesterol, purified by chromatographyjust prior to dosing. Each dose contained 8μ Ci in 15 ul 95% ethanol and40 μl Wesson oil. Two rats, the control group, were kept in the dark.One of these was killed by exsanguination after 2 hours and the otherafter 24 hours. The other two rats, the treatment group, were placedunder a sun lamp (spectral range 253-400 nm) for a total dose of 0.27joules/cm². Immediately after irradiation one rat was killed, and theother, 22 hours later. The skin, and small intestine, were removed fromeach animal and frozen on dry ice.

Individual skins were homogenized in 10 ml 0.9% saline (or in the caseof blood, enough 0.9% saline was added to give a total volume of 10 ml)and extracted with 40 ml chloroform; methanol (1:1, v/v). The chloroformphase was collected and the aqueous phase was re-extracted twice with 10ml chloroform. The chloroform phases were combined, dried under N₂ at 4°C., dissolved in n-hexane chloroform (8/2, v/v), and applied to a 1.5×30cm glass column packed with 15 grams of a Nedox-1518 Sephadex LH-20. Thecolumn was eluted with n-hexane:chloroform (8/2, v/v). Fractions (2.0ml) were collected, dried under air, dissolved in Instagel, and countedfor tritium content. With respect to the rats which received ultravioletlight, both 25-hydroxy-7-dehydrocholesterol and 25-hydroxycholecalciferol were found in major proportions in the lipid extracts ofthe skins, small intestine and blood of the rats. On the other hand,only 25-hydroxy-7-dehydrocholesterol and no 25-hydroxy-cholecalciferolwas found in the same liquid extracts from the skins. small intestinesand the blood of the rats which were kept in the dark.

EXAMPLE 4 Topical application of [24-tritiated]1,25(OH)₂-7-dehydrocholesterol on human subjects

A group of healthy volunteers of both sexes (age range of 22-30) had 1cm² area of buttock exposed to graded doses of ultraviolet irradiationfor the purpose of determining their minimal erthyema dose (M.E.D.).After determining each subject's M.E.D., 10μ Ci of[24-tritiated]-1α,25-(OH)₂ -7-dehydrocholesterol in 5-10 μl of 95%ethanol or Wesson oil was applied to a 1 cm² area on the buttock and 5minutes after application this area was either exposed to 3 M.E.D. ofultraviolet irradiation or covered to prevent any light exposure.Immediately after the ultraviolet light exposure, or an equivalent timefor the covered subjects, a 6 mm punch biopsy was obtained from the areathat received the isotope and immediately frozen on dry ice. Twenty-fourhours later 150-200 ml blood was collected.

Each skin sample was extracted with chloroform:methanol:water (2/2/0.8v/v/v) and the lipid fraction was chromatographed either on a 1.5×30 cmglass column containing 15 g Sephadex LH-20 that was slurried anddeveloped in chloroform:n-hexane (65:35 v/v) or applied directly on ahigh pressure liquid chromatograph (h.p.l.c.) equipped with achromatographic column (0.4×30 cm) killed by micro silica acid that wasdeveloped with n-hexane:isopropanol (19:1 v/v).

The blood was extracted with chloroform:methanol:blood (2:2:0.8 v/v/v)the chloroform phase was flash evaporated and the lipid residuechromatographed on a 1×60 cm glass column containing 18 g Sephadex LH-20that was slurried and developed in chloroform:n-hexane (65:35 v/v).Chromatograms of liquid extracts from the punch biopsies of the skin andblood from human subjects were carried out. Both 1α,25(OH)₂-7-dehydrocholesterol and 1α,25(OH)₂ cholecalciferol were found in theliquid extracts from the biopsies of the human skin from the subjectswhich were exposed to ultraviolet radiation. On the other hand, only1α,25(OH)₂ -7-dehydrocholesterol and no 1α25(OH)₂ -cholecaliferol weredetected in the lipid fractions obtained from the lipid extractsobtained from the biopsies from human subjects which were not exposed toultraviolet light.

EXAMPLE 5 1α,25-(OH)₂ -7-dehydrocholesterol in normal and anephric rats

A group of rats were maintained on a vitamin D-deficient diet for fourweeks. After four weeks all of the rats had their backs shaved and 24 hlater one-half of each group were bilaterally nephrectomized. Groups of6 rats received either 10 μg or 50 μg of 1α,25(OH)₂-7-dehydrocholesterol dissolved in 0.1 ml Wesson oil applied on theirbacks over a 1 cm² area while controls only had the Wesson oil vehicle(0.1 ml) applied to the backs. One hour later they were exposed to aWestinghouse sun lamp (spectral range 253-400 nm) for a total dose of0.23 joules/cm. Twenty-four hours later the animals were sacrificedtheir duodena collected for determination of intestinal calciumtransport by the everted gut sac technique and their blood was collectedfor serum calcium determination using a atomic absorption spectrometer.

The results of serum calciums of groups of vitamin D-deficient rats thatreceived a topical dose of 10 μg of 1,25(OH)₂ -7-dehydrocholesterol andthen were either exposed to ultraviolet irradiation or kept in the darkis given in the following table:

                  TABLE I                                                         ______________________________________                                        Dose 1,25(OH).sub.2 -                                                         7-dehydrocholesterol                                                                       Nephrectomy                                                                              UV       Serum Calcium                                ______________________________________                                        0                no         no     5.6 ± 0.2                               10     μg     no         no     5.7 ± 0.3                               0                no         yes    7.6 ± 0.2                               10     μg     no         yes    8.4 ± 0.2                               0                yes        no     4.6 ± 0.3                               10     μg     yes        no     4.4 ± 0.4                               10     μg     yes        yes    5.8 ± 0.2                               ______________________________________                                    

In Table I, the rats were fed a 0.47% calcium, 0.3% phosphorus vitaminD-deficient diet for 4 weeks and then some were bilaterallynephrectomized. There were 6 rats in each group and the values arereported as the mean±SEM.

The results of the efficiency of intestinal calcium transport in vitaminD-deficient anephric rats 24 hours after they had received a topicalapplication of 50 μg of 1,25(OH)₂ 7-dehydrocholesterol and then wereexposed to ultraviolet irradiation or kept in the dark is given in table2.

                  TABLE 2                                                         ______________________________________                                        Dose of 1α,25(OH).sub.2 -                                                                         Intestinal Calcium                                  7-dehydrocholesterol                                                                         UV Exposure                                                                              Transport (I/O)                                     ______________________________________                                        Control        No          1.0 ± 0.1                                       Control        Yes         1.2 ± 0.1                                       50 μg       No          1.3 ± 0.1                                       50 μg       Yes         3.1 ± 0.3                                       ______________________________________                                    

In Table 2, the rats were fed a 0.47% calcium, 0.3% phosphorus vitaminD-deficient diet for 4 weeks and then were bilaterally nephrectomized.There were 6 rats in each group and the values are reported as themean±SEM.

EXAMPLE 6

By the procedure of Example 6, the following compounds were applied tothe skins of rats at dosages of 1 μg dissolved in 20 μl of 95% ethylalcohol:

1α,25(OH)₂ D₃ ;

1α(OH)D₃ ; and

1α,24(OH)₂ 25FD₃

The control was pure ethyl alcohol.

The results of the intestinal calcium transport response and serumcalcium levels from vitamin D-deficient rats that received a topicalapplication of the above compounds, 24 hours previously, is set forth inTable 3.

                  TABLE 3                                                         ______________________________________                                        Dose           I/O        Serum Calciums                                      ______________________________________                                        Control        2.3 ± 0.1                                                                             6.3 ± 0.1                                        1α,25(OH).sub.2 D.sub.3                                                                3.8 ± 0.1                                                                             8.1 ± 0.1                                        1αOHD.sub.3                                                                            3.6 ± 0.2                                                                             8.0 ± 0.2                                        1α,24(OH).sub.2 25FD.sub.3                                                             4.2 ± 0.2                                                                             8.0 ± 0.2                                        ______________________________________                                    

EXAMPLE 7

This example demonstrates the prolonged effect of applying either1α,25(OH)₂ D₃ or 1α,25(OH)₂ pre D₃ topically.

In this experiment, rats were maintained on a Vitamin D-deficient dietadequate in calcium and phosphorus for four weeks. After four weeks, allof the rats had their backs shaved. One group of rats received 1 μg of1α,25(OH)₂ pre D₃ dissolved in 20 μl of 95% ethyl alcohol and anothergroup of rats received 1 μg of 1α,25(OH)₂ D₃ dissolved in 20 μl of 95%ethyl alcohol applied on their backs over a 1 cm² area while the controlgroup only received 20 μl of 95% ethyl alcohol applied to their backs.Another group of rats had received a dose of 1 μg of 1α,25(OH)₂ D₃ in 20μl of 95% ethanol orally administered at the same time that thiscompound was administered topically to the rats. During a period ofthirty-five days, a number from each of the group of rats weresacrificed at the same time each day. During this period, the intestinalcalcium transport and the serum calcium was measured in the same manneras set forth in Example 6 in each of the sacrificed rats. The results ofthese tests are given in FIGS. 1 through 4.

FIG. 1 is a plot of the intestinal calcium transport measured in thesacrificed rats vs. the days after administration of the dose to therats. In FIG. 1, the results of the control are compared to the resultsof rats which received either 1α,25(OH)₂ D₃ or 1α,25(OH)₂ pre D₃topically.

FIG. 2 is a plot of the serum calcium levels measured in the sacrificedrats vs. the days after administration of the dose. In FIG. 2, theresults obtained from the rats which received 1α,25(OH)₂ D₃ and1α,25(OH)₂ pre D₃ topically are set forth graphically.

FIG. 3 is a plot of the serum calcium levels measured in the sacrificedrats vs. the days after administration of the dose to the rats. In FIG.3, the results of the control and the results obtained from the ratsreceived 1α,25(OH)₂ D₃ orally are compared to the results obtained fromrats which received 1α,25(OH)₂ D₃ topically.

FIG. 4 is a plot of the intestinal calcium transport response measuredin the sacrificed rats vs. the days after administration of the dose tothe rats. In FIG. 4, the results of the control and the results obtainedfrom the rats which received 1α,25(OH)₂ D₃ orally are compared to theresults obtained from the rats which received 1α,25(OH)₂ D₃ topically.

The results in FIGS. 1 and 2 demonstrate that 1α,25(OH)₂ pre D₃ and1α,25(OH)₂ D₃ when topically applied maintained, for approximately twoweeks, an increase in both the intestinal calcium transport response andthe serum calcium level in the animals before these levels declined tothe control levels by the 21st day.

FIGS. 3 and 4 demonstrate that when 1α,25(OH)₂ D₃ is applied topicallyto the animals, the serum calcium level and the intestinal calciumtransport response were elevated for approximately two weeks afterdosing. On the other hand, when 1α,25(OH)₂ D₃ is administered orally,both serum calcium response and intestinal transport level is onlysubstantially elevated for a period of less than 7 days. Furthermore,FIGS. 3 and 4 demonstrate that when 1α,25(OH)₂ D₃ is applied orally, asharp increase in both serum calcium level and intestinal calciumtransport responses occur initially after administration, which sharpincrease does not occur when this compound is administered topically.

In examples 8 through 12, the serum ⁴⁵ Ca level in the blood stream wasmeasured by orally feeding the chickens 2 microcuries of ⁴⁵ Ca.Forty-five minutes after feeding ⁴⁵ Ca to the chickens, the chickenswere sacrificed. The serum ⁴⁵ Ca in the blood was measured by theprocedure of Haussler et al. Proc. Natl. Acad. Sci. USA, volume 70, page2248 (1973).

EXAMPLE 8

White Leghorn, one-day-old cockerels were placed on a vitaminD-deficient diet and housed under ultraviolet-free lighting. After thisperiod, 1α,25(OH)₂ -previtamin D₃ was applied topically to the comb in0.2 ml 50/50 propylene glycol/ethanol. Eighteen hours later, ⁴⁵ Caabsorption was measured. The results are given in the following table:

    ______________________________________                                        Topical Activity of 1α,25(OH).sub.2 -Previtamin D.sub.3 in              D-Deficient Chicks                                                            Topical Dose            Serum .sup.45 Ca                                      (MCG)      No. of Chicks                                                                              cpm/0.2 ml                                            ______________________________________                                        0          10           1023 ± 69                                          1          10           1151 ± 172                                         2          10           1134 ± 87                                          4          10           1704 ± 101***(+66%)                                10         10           2377 ± 131***(+132%)                               ______________________________________                                    

EXAMPLE 9

White Leghorn one-day-old cockerels were placed on a vitamin D-deficientdiet and housed under ultraviolet-free lighting. Twenty-five days later,1α,25(OH)₂ D₃ was applied to their combs topically in 0.2 ml propyleneglycol/ethanol (50/50, V/V). Eighteen hours later, serum ⁴⁵ Caabsorption was measured. The results are given in the following table:

    ______________________________________                                        Topical Activity of 1α,25(OH).sub.2 D.sub.3 in                          Vitamin D-Deficient Chicks                                                    Topical Dose             Serum .sup.45 Ca                                     (MCG)       No. of Chicks                                                                              cpm/0.2 ml                                           ______________________________________                                        0           10            438 ± 29                                         0.1         10            558 ± 45*                                                                            (+27%)                                    0.2         10            698 ± 71**                                                                           (+59%)                                    0.4         10           882 ± 147**                                                                           (+101%)                                   1           10            983 ± 119***                                                                         (+124%)                                   2           10           1221 ± 105***                                                                         (+179%)                                   4           10           1228 ± 54***                                                                          (+180%)                                   10          10           1233 ± 74***                                                                          (+182%)                                   ______________________________________                                    

EXAMPLE 10

The provitamin, 1α,25(OH)₂ -7dehydrocholesterol, dissolved in 50/50(V/V) propylene glycol/ethanol was applied topically to the combs ofchicks which had been fed a vitamin D-deficient diet for three weeks andhad been housed under ultraviolet-free lighting. Immediately afterapplication, chicks were exposed to 10 minutes under the WestinghouseFS40 Fluorescent Sun Lamps and were then returned to ultraviolet-freelighting. Eighteen hours later, the serum ⁴⁵ Ca was measured. Theresults are given in the following table:

    ______________________________________                                        Effect on UV Irradiation of D-Deficient Chicks After                          Topical Application of 1α,25(OH).sub.2 -7 Dehydrocholesterol on         Calcium Absorption                                                                 Topical Dose           Serum .sup.45 Ca                                  Exp. (MCG)      No. of Chicks                                                                             cpm/0.2 ml                                        ______________________________________                                        1     0         12           877 ± 69                                           100        12           947 ± 104                                          200        12          1120 ± 146                                          400        12          1283 ± 105**  (+46%)                                1000       12          1510 ± 123*** (+72%)                           2     0         17           730 ± 64                                           150        17           696 ± 72                                           300        16          1113 ± 125**  (+52%)                                600        14          1062 ± 101**  (+45%)                           ______________________________________                                    

EXAMPLE 11

Fifty (50) mg of 1α,25(OH)₂ -7 dehydrocholesterol were dissolved in amixture of 5 ml ethanol and 5 ml propylene glycol. The solution wasplaced in a quartz tube and suspended horizontally at a distance of onefoot under a pair of Westinghouse FS40 Fluorescent Sun Lamps. Theirradiated solution (0.2 ml) was applied topically to the combs ofchicks which had been fed a vitamin D-deficient diet and housed underultraviolet-free lighting for 3 weeks. Eighteen hours later, the serum⁴⁵ Ca absorption was measured. The results are given in the followingtable:

    ______________________________________                                        Effect of Topical Application of UV-Irradiated Solution of                    1α,25(OH).sub.2 -7 Dehydrocholesterol on Calcium Absorption in          D-Deficient Chicks                                                            UV                       Mean Serum .sup.45 Ca                                (min.)   No. of Chicks    cpm/0.2 ml                                          ______________________________________                                        0        7                686                                                 60       7               2017*** (+194%)                                      0        7                652                                                 5        7               1259**  (+93%)                                       15       7               1570*** (+141%)                                      ______________________________________                                    

EXAMPLE 12

An ointment was prepared containing the following ingredients:

    ______________________________________                                        1α,25-dihydroxy vitamin D.sub.3                                                                  2.5%                                                 Cholesterol              3.0%                                                 Stearyl Alcohol          3.0%                                                 White Wax                8.0%                                                 White Petrolatum         qs 100                                               ______________________________________                                    

Procedure:

Melt all vehicle ingredients in a suitable vessel on a water bath.Discontinue heating and add the 1α,25-dihydroxy vitamin D₃ with stirringand continue stirring until the mixture begins to congeal.

EXAMPLE 13

The ointment of Example 12 was prepared except that it contained 2.5% of1α,25-dihydroxy provitamin D₃ as the active ingredient.

EXAMPLE 14

An ointment was prepared as in Example 12 except that the activeingredient was 2.5% of 24R,25-dihydroxy provitamin D₃.

EXAMPLE 15

A hydrophilic ointment was prepared as follows:

    ______________________________________                                        1α,25-dihydroxy vitamin D.sub.3                                                                  3%                                                   Sodium Lauryl Sulfate    1%                                                   Propylene Glycol         12%                                                  Stearyl Alcohol          25%                                                  White Petrolatum         25%                                                  Methyl Paraben           0.03%                                                Propyl Paraben           0.02%                                                Deionized Water          qs 100                                               ______________________________________                                    

Procedure:

Melt the stearyl alcohol and petrolatum on a steam bath and warm toabout 75° C. Dissolve all other ingredients except the drug in the waterand warm to 75° C. Add the aqueous phase to the oil phase with stirring.Powder in the 1α,25-dihydroxy vitamin D₃ to the emulsion and stir untilsmooth.

EXAMPLE 16

A hydrophilic ointment was prepared as in Example 15 except that 5% of1α,25-dihydroxy provitamin D₃ was used as the active ingredient.

EXAMPLE 17

A hydrophilic ointment was prepared in accordance with the procedure inExample 15 except that 5% of 24R, 25-dihydroxy provitamin D₃ was theactive ingredient.

EXAMPLE 18

A cream was prepared with the following ingredients:

    ______________________________________                                        Oil Phase                                                                     1α,25-dihydroxy vitamin D.sub.3                                                                  2.5%                                                 Stearic Acid             14%                                                  Cetyl Alcohol            1%                                                   Isopropyl Palmitate      1%                                                   Propyl Paraben           0.05%                                                Sorbitan Monostearate    2%                                                   Water Phase                                                                   Methyl Paraben           0.1%                                                 Propylene Glycol         5%                                                   Polysorbate 60           1.5%                                                 Deionized Water          qs 100                                               ______________________________________                                    

Procedure:

Heat the oil phase to 70° C. Add the water phase at 72° C. to the oilphase stirring continuously.

EXAMPLE 19

A cream was prepared in the same manner as Example 18 except that 2.5%of 1 α,25-dihydroxy provitamin D₃ was used as the active ingredient.

EXAMPLE 20

A cream was prepared in the same manner as Example 18 except that 2.5%of 24R, 25-dihydroxy provitamin D₃ was used as the active ingredient.

EXAMPLE 21

A microemulsion gel was prepared with the following ingredients:

    ______________________________________                                        1α,25-dihydroxy vitamin D.sub.3                                                                  0.1%                                                 Ethoxylated Lanolin      25%                                                  Glycerine                10%                                                  Isopropyl Myristate      48%                                                  Methyl Paraben           0.1%                                                 Propyl Paraben           0.025%                                               Deionized Water          qs 100                                               ______________________________________                                    

Procedure:

The 1α,25-dihydroxy vitamin D₃, ethoxylated lanolin, isopropyl myristateand propyl paraben are heated to 80° C. The water, glycerine and methylparaben are heated to 90° C. and added to the oil phase with stirring.

EXAMPLE 22

A microemulsion gel was prepared in the same manner as Example 21 exceptthat 0.1% of 1α,25-dihydroxy provitamin D₃ was used as the activeingredient.

EXAMPLE 23

A microemulsion gel was prepared in the same manner as Example 21 exceptthat 0.1% of 1α,25-dihydroxy provitamin D₃ was used as the activeingredient.

EXAMPLE 24

Weanling male Holtzman rats were maintained on a vitamin D-deficientdiet that was adequate in calcium and phosphate for seven weeks. At theend of the seventh week on this diet, each rat had an area (2 cm²)shaved from its back. Two hours later groups of rats (6 rats each)received one of the following modes of administration in 5 to 10 μl of95% EtOH:

a. 1 μg 1α,25-dihydroxyvitamin D₃ intradermally

b. 1 μg 1α,25-dihydroxyvitamin D₃ subcutaneously

A control group received an intradermal injection of 10 μl 95% EtOH.

The results of the above experimental tests are given in the tablebelow. The intestinal calcium absorption was measured by the everted gutsac technique described by Martin and DeLuca, Arch. Biochem. Biophys.134:139-148 (1969). In the Table, the intestinal calcium transport isgiven by the ratio of the concentration of ⁴⁵ Ca outside the gut sac (0)to the concentration of ⁴⁵ Ca inside the overted gut sec (I) as setforth in the Martin and DeLuca article. The serum calcium determinationwas determined by the procedure described by Tanaka et al., Arch.Biochem. Biophys. 146:574-578 (1971).

    ______________________________________                                                          Intestinal                                                                    Calcium   Serum                                                               Absorption                                                                              Calcium Level                                     Dose              (I/O)     mg/dl                                             ______________________________________                                        10 μl 95% EtOH 1.8 ± 0.3                                                                            5.3 ± 0.1                                                        (S.E.M.)                                                    intradermally (control)                                                       1 μg 1α,25-dihydroxyvitamin D.sub.3                                                    3.4 ± 0.3                                                                            8.8 ± 0.1                                      intradermally                                                                 1 μg 1α,25-dihydroxyvitamin D.sub.3                                                    3.3 ± 0.3                                                                            8.7 ± 0.1                                      subcutaneously                                                                ______________________________________                                    

What is claimed is:
 1. A process of administering a therapeutic amount of a biologically active vitamin D material comprising providing to the skin of a subject a therapeutically effective amount of a biologically active vitamin D₂ or D₃ material of the formula: ##STR4## wherein R is methylidene or methyl; R₂ is hydrogen, hydroxy or methyl; R₁, R₃ and R₄ are hydrogen, hydroxy or halogen; A and B are individually hydrogen or taken together form a carbon to carbon bond; with the proviso that at least one of R₁, R₂, R₃ or R₄ is hydroxy and with the further proviso that when A and B form a carbon to carbon bond, R₂ is methyland allowing the material to transport through the skin into the bloodstream.
 2. The process of claim 1 wherein said vitamin D material is provided in situ to the skin by topically applying to said skin a previtamin D material of the formula: ##STR5## wherein R₁, R₂, R₃, R₄ and A and B are as above with the proviso that at least one of R₁, R₂, R₃ or R₄ is hydroxy and with the further proviso that when A and B form a carbon to carbon bond, R₂ is methyland thereafter allowing said previtamin D material to be converted to said vitamin D material through contact with said skin.
 3. The process of claim 2 wherein said 1α,24,25-trihydroxy vitamin D₃ is provided in situ to the skin by topically applying 1α,24,25-trihydroxy provitamin D₃ to the skin and thereafter subjecting said skin to ultraviolet light radiation to convert said provitamin to said vitamin.
 4. The process of claim 2 wherein said previtamin D material is 24,25-dihydroxy previtamin D₃.
 5. The process of claim 2 wherein said previtamin D is 1α-hydroxy previtamin D₃.
 6. The process of claim 2 wherein said previtamin D is 1α,25-dihydroxy previtamin D₃.
 7. The process of claim 2 wherein said vitamin D active material is provided to the skin by topically applying to the skin a provitamin D material of the formula: ##STR6## wherein R₁, R₂, R₃, R₄ and A and B are as above with the proviso that at least one of R₁, R₂, R₃ or R₄ is hydroxy; and with the further proviso that when A and B form a carbon to carbon bond, R₂ is methyland thereafter subjecting said skin to ultraviolet light radiation to convert said provitamin D material to said vitamin D material.
 8. The process of claim 7 wherein said provitamin D material is 24,25-dihdyroxy provitamin D₃.
 9. The process of claim 7 wherein said provitamin is 1α,25-dihydroxy provitamin D₃.
 10. The process of claim 7 wherein said provitamin is 1α-hydroxy provitamin D₃.
 11. The process of claim 1 wherein said material is 1α,25-dihydroxy vitamin D₃.
 12. The process of claim 11 wherein said 1α,25-dihydroxy vitamin D₃ is applied topically to the skin.
 13. The process of claim 1 wherein said material is 1α-hydroxy vitamin D₃.
 14. The process of claim 13 wherein said 1α-hydroxy vitamin D₃ is applied topically to the skin.
 15. The process of claim 1 wherein said material is 1α,24,25-trihydroxy vitamin D₃.
 16. The process of claim 15 wherein said 1α,24,25-trihydroxy vitamin D₃ is applied topically to the skin.
 17. The process of claim 15 wherein said 1α,24,25-trihydroxy vitamin D₃ is provided in situ to the skin by topically applied 1α,24,25-trihydroxy vitamin D₃ to the skin allowing said previtamin to be converted to said vitamin through contact with said skin.
 18. The process of claim 1 wherein said material is 24,25-dihydroxy vitamin D₃.
 19. The process of claim 18 wherein said 24,25-dihydroxy vitamin D₃ is applied topically to the skin. -
 20. The process of claim 1, wherein said material is 25,26-dihydroxy vitamin D₃.
 21. The process of claim 20 wherein said 25,26-dihydroxy vitamin D₃ is applied topically to the skin.
 22. The process of claim 20 wherein said 25,26-dihydroxy vitamin D₃ is provided in situ to the skin by topically applying 25,26-dihydroxy provitamin D₃ to the skin and thereafter subjecting said skin to ultraviolet light radiation to convert said provitamin to said vitamin.
 23. The process of claim 22 wherein said 25,26-dihydroxy vitamin D₃ is provided in situ to the skin by topically applying 25,26-dihydroxy previtamin D₃ to the skin and thereafter allowing said previtamin to be converted to said vitamin through contact with said skin.
 24. The process of claim 1 wherein said material is 25-hydroxy vitamin D₃.
 25. The process of claim 24 wherein said 25-hydroxy vitamin D₃ is applied topically to the skin.
 26. The process of claim 24 wherein said 25-hydroxy vitamin D₃ is provided in situ to the skin by topically applying 25-hydroxy provitamin D₃ to the skin and there with subjecting said skin to ultraviolet light radiation to convert said provitamin to said vitamin.
 27. The process of claim 24 wherein said 25-hydroxy vitamin D₃ is provided in situ to the skin by topically applying 25-hydroxy previtamin D₃ to the skin and thereafter allowing said previtamin to be converted to said vitamin D₃ through contact with said skin. 